Project Summary The general goal of the proposed research is to define the pathways and their mechanisms by which homologous recombination contributes to genome instability in somatic cells through events involving repeated DNA sequences. Using the budding yeast Saccharomyces cerevisiae as a model organism we will define the mutational signatures of multi-invasion-induced rearrangements, which are induced by a single Rad51-ssDNA filament simultaneously pairing with repeated DNA sequences on different chromosomes or locations on a single chromosome. The aims are designed to establish novel mechanisms and paradigms that are applicable to central questions concerning genomic stability and genome maintenance. We will establish novel mutational signatures caused by homologous recombination that will help to define the mechanism underlying such signatures found in humans, including cancer genomes. Moreover, our work will contribute to understanding the mechanisms underlying major processes that shape genomes during ontogenic development and evolution, including non-allelic homologous recombination, insertions, mutation showers (kataegis), double minute chromosome formation, and chromosome instability syndromes such as chromothripsis. The Specific Aims are: 1. Determine the genetic consequences of multi-invasion-induced genome rearrangements. Multi- invasion-induced rearrangements are associated with single-stranded DNA and secondary DNA double- stranded breaks. We will define the mutagenic potential of these intermediates induced by the initial single DNA double-strand break to generate secondary waves of rearrangements (Aim 1A), insertions (Aim 1B), clustered point mutations (Aim 1C), and extrachromosomal circle formation (Aim 1D). 2. Identify pathways that affect multi-invasions and the genome-wide search for homology. Using genetic endpoint analysis of multi-invasion-mediated rearrangements, we developed a mechanistic model of quality control that guards against multi-invasions. In Aim 2A, we will define the impact of the type and position of the DSB. In Aim 2B, we will use physical assays to directly quantify single- and multi-invasions as well as the DNA product to test our model. Multi-invasions are sensitive readouts of the genome-wide homology search. Paired with our physical assays, we have the unique opportunity in Aim 2C to determine to effect of global nuclear architecture and processes on the efficiency of the genome-wide homology search in vivo.